Tuberculosis is the most pervasive infectious disease world-wide, causing about 3,000,000 deaths annually. Normally associated with poverty, overcrowding and poor public health systems, the recent surge of tuberculosis cases in the United States correlates with the AIDS epidemic. The rapid progression of the disease in HIV infected persons and the increase in the number of drug resistent tuberculosis strains have highlighted the need for more rapid diagnostic methods.
The current method for rapid diagnosis of tuberculosis used in the clinical laboratory is the screening of acid-fast stained smears by microscopy. However, this method has limited sensitivity and does not differentiate M. tuberculosis from other Mycobacteria species. Liquid culture of the organisms coupled with radiometric or colorimetric detection methods offer excellent sensitivity; however, because of the slow rate of growth of Mycobacteria, it requires about 10 days for the detection of M. tuberculosis. Definitive species identification requires nucleic acid hybridization or chemical analysis of mycolic acids. (Heubner et al., Journal of Clinical Microbiology 31:771-775, 1993.) The disadvantage of direct nucleic acid hybridization assay on patient samples is that it lacks the sensitivity of the aforementioned culture methods. An alternative to amplification of the Mycobacteria by culture is the amplification of mycobacterial nucleic acid, followed by specific identification by nucleic acid hybridization. A number of nucleic acid amplification methods are being developed for mycobacterial testing including NASBA.TM. (Earle et al., American Society for Microbiology, (Las Vegas), May, 1994; van der Vliet et al., Journal of General Microbiology 139:2423-2429 (1993); and van der Vliet et al., Antimicrobial Agents and Chemotherapy 38:1959-1965 (1994), polymerase chain reaction (Eisenach et al., Journal of Infectious Diseases 161:977-981, 1990), ligase chain reaction (Iovannisci et al., Molecular and Cellular Probes 7:35-43, 1993), strand displacement (Spargo et al., Molecular and Cellular Probes 7:395-404, 1993) and Q.beta. replicase (An et al., Journal of Clinical Microbiology 33:860-867, 1995). Any of these methods could significantly reduce the time required for diagnosis of infection with M. tuberculosis, perhaps to as little as one day.
European patent application, Publication No. 0 528 306 A2 (February, 1993), discloses mycobacterium primers and probes for use in polymerase chain reaction amplification and detection. However, this publication does not show the specificity or efficiency of the disclosed primers in the polymerase chain reaction. In addition, only prophetic examples are given regarding the amplification of mycobacterial 16S rRNA. Moreover, it is accepted in the art that a pair of primers useful in a polymerase chain reaction amplification may not be useful, or as efficient, in a transcription based amplification system such as NASBA.TM..
An amplification system that has significant advantages over PCR amplification systems is the amplification system referred to as NASBA.TM. (nucleic acid sequence based amplification). The NASBA.TM. methodology is disclosed in U.S. Pat. No. 5,409,818 and European application No. 0 329 822, both of which are incorporated herein by reference. The NASBA.TM. system amplifies RNA, whereas PCR amplifies DNA. As compared to PCR, NASBA.TM. requires less user participation and fewer manipulations and steps. Another advantage is that NASBA.TM. is performed at a relatively constant ambient temperature, ensuring that the enzymes used in the process do not lose their activity. Finally, in NASBA.TM. each cycle of the amplification process generates a plurality of RNA copies from one substrate. Therefore, it is considered preferable to use the NASBA.TM. system to amplify mycobacterial RNA, which in turn can be detected using nucleic acid probes.
As in any amplification system, one must find suitable primers to amplify the sequence of interest. The need therefore exists for primer sets and hybridization probes that can be used for the amplification and subsequent detection of mycobacterium, particularly M. tuberculosis/bovis, which cause human disease. The present invention relates to the discovery of a pair of primers that give a surprisingly high yield of mycobacterial RNA, without any non-specific products, in the NASBA.TM. system.